Fig 1.
Optimization of novel serum-free condition on HeLa cells.
a, Representative images of cells in each culture condition. In screening for novel serum-free condition, 2x104 cells of HeLa cell (human cervical cancer cell line) maintained in 10% FBS/ DMEM were seeded onto each serum-free media condition. Combinations of media composition and extra-cellular matrix are shown. Improving stepwisely from RPMI-G with replacing the media composition resulted in establishment of the ITS-X/ DMEM/ BSA on fibronectin-precoated well (called as DA-X condition), which significantly improved cell adhesion, pseudopodia elongation, or proliferation. Non-coat, without any pre-coating. White and pink arrowheads indicate cells with losing attachment and cells with firm elongated pseudopodia, respectively. FN, fibronectin-coated. Scale bar, 100 μm. b, Representative images of cells cultured in DMEM/ Ham’s F-12-based medium with ITS-X/ BSA on fibronectin-precoated well. c, Representative images of cells in comparison of effect of extracellular matrix. 2x104 cells of HeLa cell maintained in 10% FBS/ DMEM were seeded onto a pre-coated well with each extracellular matrix. Regarding to cell adhesion, pseudopodia elongation, or proliferation, fibronectin was shown as much better than the other extracellular matrix. The effect of vitronectin was shown as comparable to that of fibronectin in cell adhesion and cell proliferation, but not in pseudopodia elongation. Scale bar, 100 μm.
Fig 2.
Availability of DA-X condition on analysis for cholesterol function, and in several cancer lines.
a, Effect of cholesterol biosynthesis inhibitor, Ro48-8071, on HeLa cells in serum-containing and -free culture condition for 48 hrs. In cells grown in 10% FBS/ DMEM, Ro48-8071 was shown to have no effect on cell survival and even their proliferation, while almost cells cultured in DA-X condition were shown to be dead. Scale bar, 100 μm. b and c, Comparison of the effects of RPMI-G medium and DA-X condition on cell adhesion, pseudopodia and proliferation in representative human cancer cell lines of each organ and tissue. Images of cells cultured in RPMI-G (b) and DA-X condition (c) at day 5 are shown. The number of cells at the start of culture was optimized for each cell type to reach about 80% confluency at day 5 in 10% FBS/ DMEM medium (the using cancer cell lines and their number of cells at the start of cultures in one well of a 4-well plate are shown below). HepG2 (hepatoma, 4x104), A549 (lung cancer, 2x104), Panc-1 (pancreatic cancer, 4x104), HeLa (cervical cancer, 2x104), SH-SY5Y (neuroblastoma, 4x104), SW620 (colon cancer, 4x104), PC-3 (prostatic cancer, 3x104), MCF-7 (breast cancer, 3x104). Scale bar, 100 μm.
Fig 3.
Required cholesterol level on membrane optimized by DA-X condition in HeLa cells.
a, Cholesterol labeling with filipin fluorescence staining in each culture condition. Cells were cultured in 10% FBS/ DMEM, RPMI-G, DA-X condition for 2 days, and then applied for the staining. Signals of fluorescence were enriched in membrane of cells regardless of culture condition, and the intensity was strong in 10% FBS/ DMEM and RPMI-G, but quite faint in DA-X condition. Images for filipin staining were obtained using the same exposure time. Scale bar, 50 μm. b, Quantitative PCR analysis of expression of de novo cholesterol biosynthesis-related genes. Cells cultured in each condition for 2 days, and collected for further gene expression analysis. Error bars indicate s.d. (n = 3, biological replicates). t-test, **p < 0.01 and *p < 0.05. c, Enhancement of cell adhesion and proliferation by depletion of membrane cholesterol with Methyl-β-cyclodextrin (MβCD) at 0.2 mM in RPMI-G culture medium. Pink arrowheads indicate cells with firm elongated pseudopodia. d, Effects of excess and depletion of membrane cholesterol on cell adhesion by addition of soluble cholesterol and MβCD in DA-X condition. At day 4 and 5, attenuated cell adhesion with the retraction of pseudopodia was widely observed not only in addition of cholesterol, but also in depletion of membrane cholesterol with MβCD at 1 mM. Scale bar, 100 μm. White arrowheads indicate cells with losing attachment.
Fig 4.
Involving fibronectin in down-regulation of cholesterol biosynthesis-related genes expression in DA-X condition.
a, Representative images of HeLa cells under RGDS peptide treatment in DA-X culture condition. After exposed to RGDS peptide for 3 days, retracting the pseudopodia of cells was observed accompanied by tightly association between neighbor cells. Scale bar, 100 μm. White arrowheads indicate cells with the retraction of pseudopodia. b, Quantitative PCR analysis of expression of de novo cholesterol biosynthesis-related genes. HeLa cells exposed to RGDS peptide for 4 days under DA-X condition were collected for further gene expression analysis. Error bars indicate s.d. (n = 3, biological replicates). t-test, **p < 0.01 and *p < 0.05. c, Schematic representation summarizing the appropriate range of cholesterol contents on cell membrane in serum-free medium condition for cell adhesion and proliferation.